The overall goal of this procedure is to study the protective effects of melatonin using immuno-purified primary human villous cytotrophoblast cells, cultured under hypoxia or reoxygenation. This method can help to understand the mechanisms and signalling pathways associated with pregnancy complication with placenta under hypoxia and reoxygenation. The main advantage of this technique it is suitable to study the promising treatments such as melatonin on pregnancy disorder associated with increased oxidative stress.
Generally individuals new to this method will struggle with villous cytotrophoblast isolation steps such as mincing tissues and preparation of density centrifugation median gradient. Though this matter can provide insight into third trimester trophoblast, it can also be applied to other systems such as first trimester placenta and placenta for other species. Visual demonstration of this method is important as many steps are difficult to learn without seeing them and can increases the reproducability and reduces the learning time.
Demonstrating the procedure will be graduate students Andree-Anne Hudon-Thibeault and Philippe Wong Yen and Eugenia Assuncao from my lab. To begin this procedure, cut the umbilical cord along side its placental insertion and keep them in 300 ml of tissue fixative solution for histological analysis. Then cut the entire placenta into cubes.
Wash the cubes repeatedly in saline to remove any blood cells until the saline is clear. Next, remove the placental membranes and mince the tissues to further remove blood vessels and allow calcifications. Then hold the blood vessels firmly with forceps and remove the minced tissues.
After that, place the minced placenta in a Buchner funnel and rinse the tissues with 100 ml of saline buffer. Continue mincing until 30 to 35 grams of minced tissues is obtained and place them in a weighing boat on ice. Subsequently, add the minced placental tissues to a trypsinizing flask.
Then, transfer 150 ml of the prepared digestion solution 1 to the trypsinizing flask and mix well. Place the trypsinizing flask in a shaking water bath for 30 minutes and manually mix the trypsinizing flask every five minutes for homogenizing digestion. At the end of the first digestion, remove the trypsinizing flask from the water bath and tilt it at 45 degrees for one minute to sediment the placental tissues.
With a 10 ml sterile pipette, remove and discard approximately 80 ml of supernatant without aspirating the tissue. Afterward, transfer 100 ml of digestion solution 2 to the trypsinizing flask and mix well. Place the trypsinizing flask in a shaking water bath for 30 minutes and manually mix the trypsinizing flask every five minutes for homogeneous digestion.
At the end of the second digestion remove the trypsinizing flask from the water bath and tilt it at 45 degrees for one minute. Remove 80 ml of supernatant and gently transfer it to a centrifuge tube with a cell strainer of 100 micrometer mesh. Perform the third and fourth digestions and aliquot the supernatant from digestions two, three, and four into 13.5 ml parts in the 15 ml centrifuge tubes.
Then, add 1.5 ml of FBS gently and slowly at the bottom of each tube in order to create a separate layer. Centrifuge the tubes for 20 minutes at room temperature. After that, aspirate and discard the supernatant and FBS layers, including the whitish film between the two layers.
Resuspend the pellet in 1 ml of warm cell culture medium. Collect the resuspended cells from all the tubes and combined them into one tube per digestion. Next, make up the volume to 15 ml with warm cell culture medium.
Centrifuge the cells for 10 minutes at room temperature. Afterward, remove the supernatant with a vacuum pump and avoid aspirating the pellet. Following this, gently resuspend the pellet with 1 ml of warm cell culture medium.
Pool the content of tubes of all digestions. Add additional warm cell culture medium to obtain a volume of 8 ml. Next, gently layer the cell suspension in a separation gradient and centrifuge it for 30 minutes at room temperature.
After centrifugation, identify the different layers of cells in the gradient with backlighting. Locate the layers containing trophoblasts and the contaminating cells between 40%to 50%of density centrifugation medium and remove the upper layers with a vacuum pump. Subsequently, collect the cells located in the layers of interest and transfer them to a 50 ml centrifuge tube.
Make up the volume to 50 ml with the cell culture medium and centrifuge it for 10 minutes at room temperature. Under sterile conditions, thaw the cells that were frozen in step 1.2.22 quickly in a 37-degree Celsius water bath. Transfer the thawed cells to a 50 ml tube and resuspend them gently with 20 ml of cold running buffer.
Next, centrifuge the tube for five minutes at 4 degrees Celsius. Discard the supernatant and repeat the wash step with cold running buffer. Then, count the cells using a hemocytometer to determine their viability, followed by repeating the centrifugation again.
Afterward, carefully remove the supernatant and add 1 ml of cold running buffer containing 1%volume to volume of mouse anti-HLA ABC antibody to the cells. Incubate the cells at 4 degrees Celsius for 30 minutes, mixing gently every five minutes. After incubation, add 6 ml of cold running buffer and centrifuge for 5 minutes at 4 degrees Celsius.
Discard the supernatant and repeat this step. Then, resuspend the cells in 1 ml of cold running buffer containing 10%volume to volume of anti mouse secondary antibody coupled magnetic beads. After that, incubate the cells 4 degrees Celsius for 30 minutes, mixing gently every 5 minutes again.
Subsequently, add 6 ml of cold running buffer and centrifuge for 5 minutes at 4 degrees Celsius. Discard the supernatant and resuspend the cells in 5 ml of cold running buffer. Following this, separate the trophoblast cells using the magnetic purification instrument and collect the cells at the negative port.
Add 20 ml of cold running buffer to the cells and centrifuge for 5 minutes at 4 degrees Celsius. Discard the supernatant and gently resuspend the cells in 20 ml of warm primary cell culture medium containing 10%FBS before counting the cells to determine viability. Now, plate the cells and incubate them at 37 degrees Celsius and 5%CO2.
After at least four hours, rinse the cells twice with warm culture medium to remove any unattached cells. Then, transfer the plates to the normoxia chamber, which will be filled 8%O2. Outside the hood, attach the chamber inlet port to the gas hose to reach the tube of gas.
Open both inlet and outlet ports of the chamber and keep the gas regulator closed. After that, carefully open the gas regulator valve and flush with an air flow of 25 liters per minute for 4 minutes to completely replace the air inside the chamber. After flushing the chamber, close the gas regulator, followed by the inlet and outlet ports of the chamber.
Unplug the flow meter outlet hose from the inlet port of the chamber and place the chamber in a cell culture incubator at 37 degrees Celsius for one hour. Replace the air currently present in the plates, flasks, and dissolved in the culture medium by refilling the chamber with gas at a flow rate of 25 liters per minute for 4 minutes. This figure shows the effect of melatonin on beta human chorionic gonadotropin secretion during villous trophoblast differentiation.
The biochemical differentiation of villous cytotrophoblasts into syncytiotrophoblasts was determined by the increasing values of beta human chorionic gonadotropin which reached a maximum value after 72 hours of cell culture, as shown here. Melatonin treatment had no effect on cytotrophoblast differentiation cultured in 8%oxygen compared to vehicle control. Shown here, is the effect of 1 millimolar melatonin on intercellular reactive oxygen species levels in syncytiotrophoblast cells under normoxia or hypoxia reoxygenation induced after 72 hours of culture.
Melatonin treatment protects the syncytiotrophoblasts against hypoxia reoxygenation increased oxidative stress by restoring the levels of reactive oxygen species to normoxia levels. This schema illustrates the wide intercellular protective effects of melatonin against hypoxia-reoxygenation induced oxidative stress and mitochondrial apoptosis in primary syncytiotrophoblasts. Once mastered, the isolation technique can be done in eight hours, and three hours is enough to obtain purified trophoblast cells if it is performed properly.
After its development, the technique of isolation purification, and primary cell culture of cytotrophoblasts under hypoxia-reoxygenation technique paved the way for to better understand pregnancy complications and to improve placental and fetal health. While attempting this procedure it's important to remember to evaluate the purity of villous trophoblast cells at the end of immuno-purification. For example, by flow cytometry.
After watching this video, you should have a good understanding of how to isolate an immuno-purified primary villous cytotrophoblast and culture them under hypoxia-reoxygenation.
